1. Field of Invention
The subject invention relates to a composition for the cleansing and disinfection of bones, and cleansed bone produced thereby for use in bone grafting.
2. Discussion of Background Information
A major concern in the area of bone grafting technology is the effective and safe removal of bone marrow from the less solvent-accessible cancellous bone spaces within bone grafts.
For bone grafts, human bone may be obtained from cadaveric donors under sterile conditions in an operating suite environment of local hospitals. The bone is stored frozen until it is further processed into small grafts under similar sterile conditions, or under clean-room conditions. Procurement and processing of human tissues is typically performed by groups certified by the American Association of Tissue Banks under standard operating procedures for the processing of each specific bone graft. For instance, large bones such as the femur are thawed and debrided of excess tissue prior to being cut into smaller grafts.
Processing of small bones as well as smaller bone grafts obtained from large bones includes cleaning of bone marrow from the cancellous bone spaces using mechanical means, soaking, sonication, and/or lavage with pulsatile water flow under pressure.
Bone marrow elements include hematopoietic progenitor cells, i.e., those stem cells that will ultimately differentiate into red blood cells, white blood cells, and platelets, among others. These stem cells are rich in major histocompatibility antigens (i.e., MHC antigens) that function in immune responses. It is advantageous to have bone graft material which is essentially free of residual bone marrow, for use in the preparation of small bone grafts. Large, essentially whole, bone grafts with minimal residual bone marrow offer additional advantages in that removal of bone marrow, which may harbor potential viral particles and/or viral genomes integrated into the genomes of specific cell types present in the bone marrow, reduces the potential for transmission of infective agents such as bacteria and viruses, especially the human immunodeficiency virus (HIV), since cells capable of harboring the HIV virus are abundant in bone marrow. The removal of bone marrow from large or small bone grafts also reduces the bioburden of viruses which may be present within the bone marrow cells removed.
Conventional bone-cleaning protocols may include the use of detergents, alcohol, organic solvents or similar solutes or combination of solutes designed to facilitate solubilization of the bone marrow. Common methods may use reduced or elevated temperatures, for example, between 4.degree. C. to 65.degree. C.
Ethanol and detergents have been demonstrated to be bacteriocidal toward certain bacteria, such as gonorrhea, gram negative bacteria, for example, Yersinia enterocolitica, gram positive bacteria, for example, Myobacterium tuberculosis and Chlamydia, as well as acid fast bacteria. Ethanol and detergent solutions also offer advantages of enhancing solubilization of bone marrow, reducing surface tension properties of aqueous solutions, and inactivating viruses and bacteria.
Detergents are amphiphile compounds which facilitate solubilization of relatively insoluble lipids present in, for example, bone marrow, yet at high concentrations tend to form micellar structures Helenius, A. and Simons, K., "Solubilization of Membranes by Detergents," Biochim. Biophys. Acta 415:29-79 (1975). The formation of micellar structures tends to limit the effective concentration range for detergent solutions, and thus, soaking of bone in a given volume of detergent solution may not be totally effective in that the absolute amount of detergent present is limited and if the amount of lipid material to be solubilized exceeds the solubilization capability of the detergent present, lipid solubilization will not be complete. By continually changing the detergent solution over time, it becomes possible to completely solubilize all solubilizable lipid present in bone graft.
Typically, hydrogen peroxide is used to oxidize the colored elements within the bone marrow, which results in a cleaner appearance. However, such bone often still contains bone marrow which is extremely immunogenic.
Further, most bone grafts are currently stored in the freeze-dried state. Freeze-drying removes water from the grafts, but lipid elements present in the membranes of the bone marrow cells and in vesicles present in adipocytes (i.e., fat storage cells) typically leak from the grafts after being placed in their final storage and distribution containers. These residues often give the appearance that the graft itself is not clean.
In fact, with conventional bone-cleaning protocols the graft often harbors bacteria, viruses and/or fungi in the bone marrow.
Cleaning of bone marrow from small bone grafts (for example, tarsels and meta tarsels as small as 1-5 cm) has been described in the scientific literature and in brochures and documents made public by groups involved in the procurement and processing of human tissues. A for-profit public corporation, Cryolife, Inc. (Marietta, Ga.) promotes a bone cleaning process designated as VIP.TM. (Viral Inactivation Process) and claims that the process provides "Cleaner bone through mechanical removal of debris and tissue such as bone marrow, lipids and blood components" and "Safer bone through inactivation of pathogens such as HBV and HIV (greater than 5-log kill) as well as bacteria and fungi" (Cryolife Orthopedics, Inc., brochure 12, February, 1992; Cryolife literature directed to Organ and Tissue Procurement Program Directors dated Feb. 20, 1992).
Minimal information regarding the methods of the process is available but it is described as a multi-step approach having three phases:1) preliminary surface disinfection of procured tissue for the protection of processing technicians during thawing, debriding and cutting; 2) cleaning and removal of debris from the cut pieces with a surfactant at elevated temperature; and 3) terminal disinfection of the cleaned bone grafts (The Viricidal Capacity of a Surfactant/Iodophor-Based Viral Inactivation Process for Bone Allografts, Cryolife documentation). The VIP process is claimed to both clean bone allografts, e.g., a femur head, and to inactivate a variety of bacteria and viruses without affecting bone strength or biological properties. However, according to documents made public by Cryolife, Inc., the process is used to clean the surfaces of large bone grafts and to remove bone marrow from the cancellous bone spaces of small bone grafts cut from the larger grafts.
A second, for-profit publicly held corporation, Osteotech, Inc., Shrewbury, N.J., describes a bone graft cleaning process called Permein ("a combination of ethanol and non-ionic detergent"; Mellonig, J. T., Prewett, A. B., and Moyer, M. P., J. Periodontal 63:979-983 (December, 1992). This Process involves the use of a solution of ethanol and detergent to clean bone grafts. Details of the process and detergents utilized are not currently available. Bone is soaked in the solution and it is claimed that the combination of ethanol and detergent facilitates permeation of the solution into bone. The process has been demonstrated to clean small cut-bone grafts and to be capable of inactivating the HIV in bone allograft (finely ground bone) (Mellonig, Prewett, and Moyer, J. Periodontology:979-983 (December, 1992).